Hematuria is the most common presentation of bladder cancer (BCa) with 22% of patients with gross hematuria and 8% of patients with microscopic hematuria harboring BCa. Voided urinary cytology (VUC) is the most widely used urine-based assay (and gold standard) for detecting BCa; however, it fails to detect approximately 50% of low-grade or early stage BCa when it is most curable. Furthermore, the detection rate of VUC for recurrent BCa is not much better. Because of this severe limitation, all patients with hematuria and who are under surveillance to monitor for recurrent BCa must undergo an invasive examination of the urinary bladder, where a miniature camera is inserted into the bladder and the bladder inspected. We propose to improve the detection of BCa by utilizing a multiplex ELISA for the detection of a previously validated BCa- associated diagnostic signature (signature) in voided urine. Hypothesis A molecular profile exists that a) is specificall associated with BCa and b) can be utilized to indicate the presence of BCa in non-invasively obtained voided urine samples. This hypothesis will be tested by completing the following Specific Aims: 1) To validate the multiplex ELISA for BCa detection in subjects presenting with gross hematuria, 2) To validate the multiplex ELISA for BCa detection in patients with a history of BCa currently undergoing tumor surveillance and 3) To investigate the ability of the BCa-associated diagnostic signature to determine risk of disease. Significance This research will open the door for improving on the non-invasive methods for detecting BCa and will have a marked impact on patient care. Methodology Our group has developed and tested a multiplex ELISA towards this signature. In this proposal, we will test the multiplex ELISA in 2 independent studies: patients presenting with gross hematuria and BCa patients undergoing tumor surveillance. Expected Results The validation of the multiplex ELISA will reduce the need to subject large numbers of patients who do not have BCa to frequent, uncomfortable and expensive cystoscopic examinations. Specifically, we would anticipate a reduction in the number of cystoscopies/year by 500,000, which would translate into cost savings of $225 million/year.